3.7. CTRP9 Overexpression Enhances Features of Atherosclerotic Plaque Stability
In order to verify whether CTRP9 has a protective effect on atherosclerosis, adeno-associated virus was used to construct an animal model of CTRP9 overexpression. The western blotting result showed that compared with AAV-GFP, AAV-CTRP9 significantly increased CTRP9 expression in Apoe-/-mice (Fig. 7e). To examine the effects of CTRP9 on atherosclerotic plaque stability, we first analyzed the lesion sizes using HE staining. Consistent with our previous study findings, CTRP9 reduced atherosclerotic plaque size (Fig. 7a). To determine whether CTRP9 overexpression influences the features of plaque stability or not, we performed Sirius Red staining to determine plaque collagen level. The plaque collagen level, which plays an important structural role in stabilizing plaques, was increased in CTRP9-overexpressed mice relative to that in the control mice (Fig. 7b). Additionally, the number of MYH11-positive cells was increased within plaques from CTRP9-overexpressing mice relative to those from the controls, and this is also consistent with increased plaque stability (Fig. 7c). Finally, the expression of RIP3 was significantly decreased in plaque in CTRP9-overexpressed mice relative to those from the control mice (Fig. 7d). Western blot was used to detect the expression of markers related to necroptosis in mouse aorta. The results showed that overexpression of CTRP9 significantly reduced the levels of p-RIP1, RIP3 and p-MLKL compared with the control group (Fig.7e and f), which indicates that the number of programmed necrotic cells in atherosclerotic plaque is significantly reduced. In addition, we also detected the expression levels of various inflammatory factors in the aorta. The results showed that the expression of inflammatory factors in the aorta of CTRP9-overexpressed mice was significantly decreased (Fig.7g). Overall, these results demonstrate that CTRP9 promotes features of a stable plaque phenotype in atherosclerosis.